CN102872463A - Nanoparticles comprising rna ligands - Google Patents

Abstract

Materials and methods are provided for making nanoparticles having a core including metal and/or semiconductor atoms. The core is covalently linked to a plurality of ligands comprising a RNA ligand. The RNA ligands may include siRNA or miRNA. Also provided are uses of these nanoparticles in therapy and diagnosis.

Description

The nano-particle that contains the RNA part

It is 201207160023797.0 that the application of this division is based on application number, and the applying date is on 05 24th, 2005, and denomination of invention is divided an application for the Chinese patent application of " nano-particle that contains the RNA part ".

Technical field

The present invention relates to nano-particle, relate more specifically to contain the nano-particle of RNA part such as siRNA (siRNA) and microRNA (miRNA), and the purposes in various application.

Background technology

Have been found that small RNA molecular plays multiple effect in regulator gene is expressed.These effects comprise siRNA (siRNA) to the orientation degraded of mRNA, PTGS (PTG), and microRNA (miRNA) is to growth controllable type sequence-specific translation inhibitory action and the directed transcriptional gene silencing of mRNA.RNA i activity has limited transposon and has shifted and provide antiviral defense (Pal-Bhadra etc., 2004).Also verified RNAi mechanism and little RNA are in the targeting of heterochromatin complex and the effect (Verdel etc., 2004) in the epigenetic gene silencing on the specific chromosomal foci.Reticent behind double-stranded RNA (dsRNA) dependent transcription, be also referred to as little inhibitions RNA(siRNA) or RNA disturb (RNAi), be wherein the dsRNA complex can the accurate specific homologous genes of targeting with the phenomenon of silence in short time period.It has the signal of the mRNA degraded of sequence homogeneity as promotion.The common long enough of 20-nt siRNA is reticent with the induced gene specificity, and enough short in to avoid host response (Elbashir etc., 2001).The reduction of directed gene Product Expression can be on a large scale, has 90% silence of being induced by several siRNA molecules.

Because sending of micromolecule oligonucleotide can be walked around the difficulty relevant with gene therapy, so use siRNA may have the advantage that is better than traditional gene therapy.Up to now, effectively send obstacle in the gene therapy that remains successful in the body based on the therapeutic gene of carrier.Although having observed by siRNA is not that nonvolatil, independent siRNA transfection can cause the inhibition of target protein in parent and the daughter cell to prolong (Tuschl, 2001) to the rejecting of target gene.Yet sending in the art of siRNA still has problems.

WO 02/32404(Consejo Superior de Investigaciones Scientificas) disclose the nano-particle that is formed by metal or semiconductor atom, the core that wherein comprises the part of carbohydrate and nano-particle is covalently bound.These nano-particle are used for regulating the interaction of carbohydrate mediation and are solvable and nontoxic.Enjoy GB-A-0313259.4(Consejo Superior de Investigaciones Scientificas and Midatech Limited) PCT of priority application discloses the magnetic nanoparticle with core, this core comprises the magnetic metal atom of passive state, and core and part are covalently bound.

Summary of the invention

Loosely say, the present invention relates to have the nano-particle of core, this core comprises metal and/or semiconductor atom, and core and RNA part are covalently bound.The RNA part normally designs to simulate the short rna sequence of siRNA (siRNA) and microRNA (miRNA) sequence.Nano-particle can be used for sending the RNA part and having widely purposes, can use in vitro system and be used for the treatment of or diagnose.For example, nano-particle of the present invention can be used for (1) directed transcriptional gene silencing, (2) directed mRNA degraded, (3) mRNA imaging, (4) by suppressing various approach with the multiple RNA part on the identical or different nano-particle, (5) aerosol is sent, and for example to lung, (6) are used as the instrument of functional genome in conjunction with the reticent targeting siRNA resistance mRNA of being used for of mRNA and (7).

In the art, according to its source the short rna sequence is called " short interfering rna " (siRNA) or " microRNA " (miRNA).Two types sequence can be by in conjunction with complementary RNA (nmRNA) with cause mRNA and eliminate (RNAi) or stop mRNA to translate into protein and be used for down-regulation of gene expression.SiRNA produces by the processing of long dsrna, and external source normally when in fact finding.Little RNA interfering (miRNA) is the little non-coding RNA of interior source code, and the processing of pressing from both sides by bob produces.SiRNA and miRNA can suppress do not have RNA to shear with the translation of the mRNA of part complementary target sequence, and can degrade with the mRNA of fully-complementary sequence.The RNAi approach also acts on genome, as is described in Science, 301:1060-1061,2003.

The RNA that links to each other with nano-particle can be strand or double-stranded (duplex).As in the situation of part, the RNA sequence can be hair clip with miRNA sample sequence, namely comprises the complementary district of part, and can anneal near them forms the end of hair clip.Nano-particle can randomly comprise the part of other type, forms sugared nano-particle such as carbohydrate, and/or more than a kind of siRNA.Below will discuss nano-particle and uses thereof in more detail.Advantageously, siRNA makes it not to be subjected in blood, the tissue culture medium (TCM) or the impact of the exoribonuclease that exists in the cell with the protection on siRNA can be provided being connected of nano-particle.

Therefore, in first aspect, the invention provides the nano-particle that contains core, this core comprises metal and/or semiconductor atom, and wherein covalently bound the and part of core and multiple ligands comprises the RNA part.

The siRNA part that forms nano-particle can be strand or double-stranded (duplex).Yet, effectiveness for the target gene function downward modulation of optimization RNA mediation, preferably select the length of siRNA molecule to guarantee that the RISC complex is to the correct identification of siRNA, when the compound-mediated mRNA target of described RISC was used nano-particle in the identification of siRNA and the preferred body, siRNA is the short host response that reduces enough.

The miRNA part normally strand and have a complementary district of the part that can make part form hair clip.MiRNA is the form of single stranded RNA normally, and thinks the expression of regulating other genes.MiRNA transcribes from DNA, but does not translate into the rna gene of protein.The DNA sequence of coding miRNA gene is longer than miRNA.This DNA sequence comprises that miRNA sequence and approximate reverse are to complement.Divide the period of the day from 11 p.m. to 1 a.m when this DNA sequence is transcribed into single stranded RNA, miRNA sequence and reverse mutual complement base pair thereof form the double-stranded RNA sections; Generally speaking, the structure of this RNA is called hairpin structure (' short hairpin RNA ' or shRNA).Then the Dicer enzyme cuts away double stranded region from hairpin structure, and discharges ripe miRNA.

By under the control of rna plymerase iii promoter such as people H1 or 7SK promoter, using the DNA construct transfectional cell of coding shRNA sequence, can in cell, produce shRNA.Perhaps, can externally synthesize shRNA also directly introduces in the cell.

Usually, the RNA part that is intended to simulate siRNA and miRNA effect has 10 to 40 ribonucleotides (or its synthetic analogues), more preferably 17 to 30 ribonucleotides, more preferably 19 to 25 ribonucleotides, most preferably 21 to 23 ribonucleotides.In embodiments more of the present invention of using double-stranded siRNA, this molecule can have 3 ' symmetrical jag, for example, and the 3 ' jag of one or two (ribose) nucleotide, the normally UU3 ' jag of dTdT.

Produce in the situation of mi RNA in the shearing by shRNA, 40 to 100 bases of shRNA sequence preference are long, and more preferably 40 to 70 bases are long.Preferred 19 to 30 base pairs in the stem district of hair clip are long.The stem district can contain G-U and match to stablize hairpin structure.

In using the embodiment of double-stranded RNA, the sense and antisense chain formation duplex of can annealing.Form nano-particle by comprise duplex in reactant mixture, RNA can be connected to core in the process that granule assembles automatically.Quantity according to double-stranded siRNA derivation terminal (four 5 ' and 3 ' possible ends of every chain), four nano-particle can be connected on the single siRNA duplex at the most, and for each double-stranded siRNA, be formed in theory many 15 possible constructs, one in these has four nano-particle (each terminal two), four have a nano-particle, and six have two nano-particle and four and have three nano-particle.In the situation of using strand siRNA, the nano-particle core can be connected on any or two the derivation ends (that is, 5 ' or 3 ' end) of siRNA, for example, produces three kinds of different nano-particle.These nano-particle can use or the method can randomly comprise the complementary strand annealing that makes the siRNA that contains nano-particle and siRNA with this form, thereby original position forms the other step of duplex on preformed nano-particle.In the forming process of miRNA sample part, one or two nano-particle is connected on the end of RNA sequence usually.

Therefore, in another aspect, the invention provides the production method of said nano-particle.Usually, the method comprises puts together the core of RNA part and nano-particle, by with junctional complex derivation RNA chain and the RNA of derivation is contained in the reactant mixture of synthesis of nano granular core thus.In the process that nano-particle assembles automatically, the nano-particle core is connected on the RNA by junctional complex.Preferably, junctional complex is disulfide linkers, and the disulfide linkers that for example mixes is although also can use ethylidene junctional complex or peptide junctional complex.The example linking group is by general formula HO-(CH ₂) _n-S-S-(CH ₂) _m-OH represents, wherein n and m are 1 to 5 independently.RNA, by a terminal hydroxyl, can be connected with sept in the situation of preferred mixed disulfide junctional complex easily by the terminal phosphate group.When the synthesis of nano granule, junctional complex-S-S-breaks to form two sulfur-bearing junctional complexs, separately by-the S-group can be covalently bound to the core of nano-particle.Use the disulfide linkers that mixes to help avoid the formation of RNA dimer.

As mentioned above, be in the situation of strand at the RNA that is connected with the nano-particle core, the method can comprise makes the other step of annealing to provide the double-stranded RNA part that is connected on the nano-particle with the RNA molecule of the first chain complementation.The nano-particle that can also prepare the double-stranded RNA with annealing, one of this RNA or two chains are by the disulfide linkers functionalization.Alternatively or additionally, the sense and antisense chain of this RNA can be connected on the different nano-particle and together annealing.

In the preferred embodiments of the invention, for double-stranded RNA being mixed in the nano-particle in the automatic assembling, can come with the disulphide that mixes one or two end of this RNA of derivation.After mixing duplex in the nano-particle, the chemical composition of RNA and mixed disulfide is all mixed in the beadlet.Therefore, the chemical composition of mixed disulfide can comprise important information (for example, the member that specific binding is right) such as the targeting characteristic with the character of itself, other physical characteristic is provided maybe might for the final nano-particle that forms.The disulphide that mixes can be connected on the 3 ' end or 5 ' end of sense or antisense chain.

In one embodiment, use and can synthesize the nano-particle with core in the method described in the WO 02/32404 first, this core comprises gold atom, wherein comes the derivation part with disulfide linkers and make part and the HAuCl of derivation in the presence of Reducing agent ₄(tetra chlorauric acid) reaction generates nano-particle.According to the method, the RNA of the protection of the disulphide in methanol or the water can be added in the aqueous solution of tetra chlorauric acid.Preferred Reducing agent is sodium borohydride.These and other feature descriptions of the method are in WO 02/32404.

Some can use many different RNA molecules in using.Can be used as the different ligands of puting together from one group of nano-particle, these different RNA molecules or different RNA molecules are provided can be independent nano-particle group, and randomly mixes.In the first situation, those skilled in the art will recognize that by RNA and nano-particle and put together in the situation that forms product mixture, one group of nano-particle can comprise multiple aforesaid product.In the situation of using a plurality of parts, can use the part of simulation siRNA and miRNA.

Except the RNA molecule, part and/or RNA part that nano-particle can comprise one or more other types can also comprise one or more dissimilar group or domains except the RNA component.For example, other part, or the group of part or domain can comprise one or more peptides, protein domain, nucleic acid molecules, lipid groups, carbohydrate group, any organic or anion or cation group.Carbohydrate group can be polysaccharide, oligosaccharide or monosaccharide group.Preferred part comprises glycoconjugate, thereby forms sugared nano-particle.As following pointed in the discussion of nano-particle purposes, part (RNA or other part) can be the right member of specific binding and the target position that is used for the nano-particle targeting is wherein existed another right member of specific binding.Also exist except the RNA part in the situation of nucleic acid molecules, this nucleic acid molecules can comprise list or double-stranded DNA or RNA.Granule can have the part that is fixed thereon more than a kind of, for example, and 2,3,4,5,10,20 or 100 kind of different ligands.Alternatively or additionally, can use together the nano-particle of number of different types.In the preferred embodiment, the average that is connected to the whole parts on the granule single metal core is at least one part, more preferably 50 parts, most preferably 60 parts.

Preferably, it is 0.5 to 50nm that nano-particle has average diameter, and more preferably 0.5 to 10nm, and more preferably 1.0 to 5nm, more more preferably 3.0 to 7.0nm core.When also considering part except core, the grand mean diameter of preferred particulates is 5.0 to 100nm, and more preferably 5 to 50nm, and most preferably 10 to 30nm.Can measure average diameter with technology well known in the art such as transmission electron microscopy.

Core material can be metal or quasiconductor and can be formed by the atom more than a type.Preferably, core material is to be selected from Au, the metal of Fe or Cu.The nano-particle core can also be formed by alloy, and described alloy comprises Au/Fe, Au/Cu, and Au/Gd, Au/Fe/Cu, Au/Fe/Gd and Au/Fe/Cu/Gd, and can be used for the present invention.Preferred core material is Au and Fe, and most preferred material is Au.The core of nano-particle preferably comprises approximately, and 100 to 500 atoms (for example, gold atom) provide nano level core diameter.The atom of one or more NMR activity of mixing in other useful especially core materials is so that can detect nano-particle with NMR in vitro and in vivo.The example of NMR active atomic comprises Mn ^{+ 2}, Gd ^{+ 3}, Eu ^{+ 2}, Cu ^{+ 2}, V ^{+ 2}, Co ^{+ 2}, Ni ^{+ 2}, Fe ^{+ 2}, Fe ^{+ 3}And lanthanide series ^{+ 3}, or the described quantum dot in the application other places.

Can detect the nano-particle core that contains semiconductor atom, because the nano semiconductor crystal can be as quantum dot, be that they can absorbing light, thus with the electron excitation in the material to higher energy level, discharge subsequently the photon of light with the characteristic frequency of material.The example of semiconductive core material is cadmium selenide, cadmium sulfide, cadmium telluride.Also comprise zinc compound, such as zinc sulfide.

In some embodiments, nano-particle of the present invention or RNA molecule comprise detectable labelling.This labelling can be the composition of nano-particle core or RNA part or another kind of part.But because the inherent character of this composition of nano-particle or by being connected with the another kind test section, put together or continuous, this labelling is detectable.Preferred labelling example is included as fluorophor, radionuclide, the labelling of magnetic mark or dyestuff.Fluorophor comprises fluorescein, rhodamine or tetramethylrhodamin, Texas-is red, Cy3, Cy5 etc. can detect (Y.C.Cao by the fluorescently-labeled light that excites and launch with the detection of raman scattering spectrum algoscopy, R.Jin, C.A.Mirkin, Science 2002,297:1536-1539).

In some embodiments, nano-particle can comprise radionuclide, is used for detecting nano-particle with the radioactivity of radionuclide emission, for example, uses PET, SPECT, or be used for the treatment of, namely be used for kill target cell.Commonly used being easy to adjusted and comprised to be suitable for radionuclide example of the present invention in this area ^99mTC, it exists with multiple oxidation state, although the most stable be TcO ^{4- 32}P or ³³P; ⁵⁷Co; ⁵⁹Fe; Usually with Cu ²⁺Salt uses ⁶⁷Cu; Usually with Ga ³⁺Salt uses ⁶⁷Ga, for example, gallium citrate; ⁶⁸Ge; ⁸²Sr; ⁹⁹Mo; ¹⁰³Pd; Usually with In ³⁺The 111In that salt uses; Usually use with sodium iodide ¹²⁵I or ¹³¹I; ¹³⁷Cs; ¹⁵³Gd; ¹⁵³Sm; ¹⁵⁸Au; ¹⁸⁶Re; Usually with Tl ⁺Salt uses such as thallium chloride ²⁰¹Tl; ³⁹Y ^{3+ 71}Lu ³⁺With ²⁴Cr ²⁺Radionuclide is with marking and the general service of tracer is well known in the art and those skilled in the art can easily adjust to be suitable in the each aspect of the present invention.By it being doped in the core of nano-particle or comprising that they as the labelling that a part part that is fixed on the nano-particle exists, can use radionuclide easily.

Additionally or replacedly, can use multiple technologies well known in the art, detect nano-particle of the present invention with the aforesaid labelling that links to each other with nano-particle or by the characteristic with them, or the result of they and other component interaction.The gathering that these methods that detect nano-particle can occur when detecting nano-particle in conjunction with another kind of component, for example, by simple range estimation or use light scattering (absorbance that contains nanoparticles solution), to using sophisticated technology such as transmission electron microscopy (TEM) or atomic force microscopy (AFM) to observe nano-particle.The other method that detects metallic particles is to use plasmon resonance, namely in the electron excitation of metal surface, is usually caused by rayed.Surface plasmon resonance (SPR) phenomenon is present on the interface of metal (such as Ag or Au) and insulant such as air or water.Along with analyte is bonded to the refractive index that the part that is fixed on the nano grain surface has changed the interface, change has occured in SPR.Another advantage of SPR is can be used for monitoring to interact in real time.As mentioned above, if nano-particle comprises or the NMR active atomic that mixed, this technology can be used for using technology well known in the art to detect this granule in external or body so.Can also use the system of amplifying based on quantitative signal, the silver (I) that utilizes nano-particle to promote also detected nano-particle originally.If nano-particle comprises the part as fluorescent probe, then can use spectrofluorometry.In addition, the isotopic labeling of carbohydrate can be with the detection of helping them.

The invention provides the method for the ball array that presents part, this array has the advantage of the other types array that is better than proposing in the prior art.Especially, nano-particle especially is soluble in water in most of organic solvents.This can be used for their purification and importantly mean and can use in solution, is used for presenting the part that is fixed in particle surface.The fact is that soluble nano-particle has the advantage that presents the native conformation part.Use for treatment, nano-particle is nontoxic under physiological condition, and is soluble and be stable.

In some embodiments, the core of nano-particle can be magnetic and comprise the magnetic metal atom, randomly be combined with the passive metal atom.For example, passive metal can be gold and platinum, silver or copper, and magnetic metal can be ferrum or gadolinium.In the preferred embodiment, passive metal is that gold and magnetic metal are ferrum.In this case, in the core proper proportion of passive metal atom and magnetic metal atom be approximately 5:0.1 to about 2:5.More preferably, ratio be approximately 5:0.1 to about 5:1.As used herein, term " passive metal " refers to and does not demonstrate magnetic properties and be chemically stable metal for oxidation.Passive metal of the present invention can be diamagnetic.Diamagnetism refers to the material with whole paired electronss, so its each atom does not have permanent Net magnetic moment.Magnetic material have some not paired electronics and for the external magnetic field be absolute responsive-namely, the external magnetic field induces electronics along with externally-applied magnetic field is lined up, so electronic magnetic moment is adjusted.Magnetic material can be paramagnetic, and is superparamagnetism or ferromagnetic.Paramagnetic material is not very responsive to the external magnetic field, and no longer keeps their magnetic properties when removing the external magnetic field.Ferrimagnet is extremely sensitive to the external magnetic field, even also contains magnetic domain when not having the external magnetic field to exist, because contiguous atom cooperation is so that their electron spin is parallel.The magnetic moment of adjacent domain is adjusted in the external magnetic field, has enlarged magnetic effect.Usually the very little granule that has the ferromagnetic characteristics material is not ferromagnetic, because do not produce cooperative effect in 300nm or less granule, so that material does not have permanent magnetic.Yet granule is still very responsive and have strong paramagnetism characteristic to the external magnetic field, is called superparamagnetism.Preferably, nano-particle of the present invention is superparamagnetism.

Example with the nano-particle that comprises the paramagnetic metal core comprises and contains Mn ^{+ 2}, Gd ^{+ 3}, Eu ^{+ 2}, Cu ^{+ 2}, V ^{+ 2}, Co ^{+ 2}, Ni ^{+ 2}, Fe ^{+ 2}, Fe ^{+ 3}And lanthanide series ^{+ 3}Those.

Can be from material such as the MnFe(ferrospinel that can form nano-particle (magnetic fluid)) or the CoFe(Conjugate ferrite) form other magnetic nanoparticles, add or do not add other aforesaid core material.Biotechnol.Prog., 19:1095-100(2003), J.Am.Chem.Soc.125:9828-33(2003), provided the example for the production of the automatic assembly connection chemistry of such nano-particle J.Colloid Interface Sci.255:293-8(2002).

On the other hand, the invention provides compositions, comprise the group of one or more above institutes definitions particles.In some embodiments, the nano-particle group can have the identical or different part that is connected in core of different densities.In the certain situation, it is desirable to nano-particle is incapsulated a plurality of nano-particle can be delivered to target site.Suitable encapsulated technology is well known to a person skilled in the art.Encapsulated nano-particle group can be a kind of, two kinds, and three kinds or multiple different type.In one embodiment, the invention provides the aerosol combination at this defined nano-particle.Aerosol combination can comprise nano-particle and optional diluent.The example of these compositions purposes below is discussed.

Unrestriced mode provides the example of following nano-particle application to support the broad applicability of said technology by explanation.Dorseet ﹠amp; Tuschl, Nature Reviews, 3:318-329 provides the general purpose summary of siRNA in 2004.

On the other hand, the nano-particle that the invention provides above definition be used for the treatment of or diagnose in purposes.

On the other hand, the invention provides the nano-particle of above definition for the preparation of the purposes of medicine, this medicine is used for the treatment of the disease that is eased by giving nano-particle.Below described according to the present invention the example of treatable concrete purposes, other application with nano-particle comprise the purposes in external and the body.For example, nano-particle or derivatives thereof described herein can be formulated in the pharmaceutical composition, and delivers medicine to the patient with various forms, especially for treating the disease of being alleviated by giving the RNA part.For example, this can be used for the treatment of the disease of alleviating by by the expression of RNA down-regulated gene, wherein comes down-regulated gene by RNA, or is used for the treatment of the disease relevant with the overexpression of the gene of also reducing by the RNA targeting.

Enhancer elements

The nano-particle that comprises the RNA part can be used for regulator gene in many ways expresses, comprise the orientation degraded by siRNA (siRNA) mRNA, PTGS (PTG), by little-RNA(miRNA) the growth controllable type sequence-specific translation of mRNA suppresses and directed transcriptional gene silencing.

Generally speaking, the invention provides the purposes that nano-particle described herein is used for the downward modulation target gene.Among the application, downward modulation can be external, for example, study gene expression, or in the body, in the target experimental system or be used for medical usage, namely nano-particle can be used for preparing the medicine of disease that treatment alleviates by the expression of RNA downward modulation target gene or the treatment disease relevant with the target gene overexpression.For example, this disease can comprise cancer, for example, breast carcinoma, it can use the RNA based on the Her2/Neu sequence to treat.As described herein, because in fact the downward modulation that is provided by RNA may be temporary transient, so, in some embodiments, preferred nano-particle further comprises radionuclide, and medicine or other medicaments, described medicament are used for the treatment of or kill the wherein cell of nano-particle downward modulation target gene.

RNA disturbs the disease that can be used for the treatment of any with too active gene-correlation, and for example the cancer of most of forms for example, is used RNA to be used for oncogene and suppressed.Specific gene such as hepatitis or too active gene expression help that shutting down of cell death receptor also is the target of RNA treatment in other diseases of nosopathology.The other example that uses the suitable disease of nano-particle treatment of the present invention is the degeneration of macula in the eyes, or utilize the natural action conduct of RNA to lose the method that ability is resisted pathogenic virus by the RNA that makes virus, these viruses are particularly including HIV, hepatitis C or influenza (Check, 2003; Zamore etc., 2003; Song etc., 2003; Matzke ﹠amp; Matzke, 2003).

The downward modulation of approach

It should be noted that especially the present invention allows to send the RNA molecule more than a kind of, this is impossible thing in the past in the art always.Therefore, in some embodiments, the invention provides the Nanoparticulate compositions that contains at least two kinds of different RNA sequences, this RNA sequence and identical nano-particle are puted together or are present in the compositions of at least two kinds of dissimilar nano-particle, can be used for expression in the down-regulated gene approach.The number of the RNA part that exists in the compositions will depend on the complexity of approach and the number gene that needs targeting to be used to reduce.The example of approach that can targeting is used for research or treatment, comprises the approach that causes inflammation, antiviral pathway, the signal pathway in the cancer, route of metastasis or metabolic pathway.For example, this comprises the adjusting of the new sugared generation approach that produces for glucose in the type ii diabetes.

In another relevant embodiment, come domain conservative between the targeting family member by designated rna, the RNA-nano-particle can be used for the expression of down-regulated gene family.

Any purposes that is used for using the RNA inhibition of gene expression at nano-particle, nano-particle can also comprise siRNA sequence and the reticent sequence of mRNA is come targeting mRNA.These nano-particle can be used for suppressing siRNA resistance mRNA.This can be used for wherein that target cell is the situation of resistance to the siRNA silence, because their express the protein that blocking-up siRNA suppresses mechanism.In this case, can express the gene outcome of induction of resistance to improve the siRNA resistance by siRNA is aimed at.

Use other parts or use the targeting of RNA to use

In a kind of application, Nanoparticulate compositions of the present invention can be used for giving the targeting characteristic, is used for RNA is delivered to target cell.This can by provide have an other type with close the part that the nano-particle core puts together or the nano-particle that can make RNA nano-particle and the interactional domain of target cell group specificity that links to each other with the RNA part and realize.For example, the nano-particle that the contains RNA cell mass that can preferentially lead, by the nano-particle with part is provided, this part is the right member of particular combination who can specific binding be present in target cell surface or inner binding partners, for example, targeting specific cells structure is such as nuclear.The right example of particular combination that is suitable for use as the part of puting together with the nano-particle that is used for targeting comprises part and receptor, and many alternatives are that those skilled in the art are apparent.For example, the nano-particle of glucose derivation can be used for the cell (18) that targeting contains protein G LUT family member.Can use other the carbohydrate ligands that is used for nano-particle, such as Glc β 4GlcNAc or Glc β 4GlcNH ₂The former can be used at first will containing the GLUT transport protein on the nano-particle targeted cells surface of siRNA, and then when entering cell (after glucosidase is sheared), GlcNAc will further examine siRNA nano-particle targeting.A rear construct is added into positive charge on the surface of nano-particle, further promotes to adhere to and absorb to cell.Because the nano-particle of assembling can be used for mixing allos part such as lipid automatically, peptide or any other chemical composition are (for example, described in as above part is discussed), it is connected with disulphide by sept, and multiple other targeted moleculars can be used for the specific cell type of RNA nano-particle targeting.For example, these technology can be used for carrying the nano-particle target tumor cell of RNA.

Nano-particle is used for another example of targeted cells type purposes, and the RNA part of nano-particle can be as the entity that the nano-particle targeting is provided, the cell that the nano-particle guiding is wherein expressed with the mRNA of RNA ligand interaction.The example of the target cell type of targeting is the cell of tumor cell or viral infection by this way, comes the expression of viral gene in the targeting target cell or tumor marker or oncogene with RNA.In this embodiment, preferred RNA-nano-particle can permeate through cell membranes, this be provided by their small size and a kind of effect (referring to Nature Biotechnology 18:410-414,2000) by strengthening with film transposition signal derivation nano-particle randomly.The RNA-nano-particle has the advantage of reducing said target mrna in the cell selective mode to the targeting of the cell of wherein expressing corresponding mRNA.Yet, because this effect is normally temporary transient, thus the nano-particle with radionuclide or medicine preferably is provided, so that use the cell of RNA targeting to be killed by selectivity.Can with target cell and processing procedure imaging, follow marked with nanometer granule, for example nano-particle of aforesaid use radioactivity or magnetic.

Make the mRNA imaging

On the other hand, the invention provides the method for mRNA detection and/or imaging, it has used nano-particle described herein.Especially, before the present invention, there is not the known mRNA image formation method that makes in the prior art.The method can be included in and make in the body under the RNA part that wherein is present on the nano-particle and the interactional condition of said target mrna or the RNA contact nanometer granule in the sample, and detects nano-particle-RNA-mRNA complex.Detect the step of complex and can use the inherent character of nano-particle or the labelling that links to each other with nano-particle by detection.The preferred embodiment that is applicable to the labelling in this respect of the present invention comprises the nano-particle that contains the group that is magnetic, quantum dot or radionuclide.Quantum dot, for example, the nanocrystal by cadmium selenide provides, or can also use other aniones such as sulfide except selenides, and it has potential purposes in the screening of biological imaging, electronics and optical device, quantum computer and drug candidate.

The RNA-nano-particle is as the instrument of functional genome

Genome screening utilizes the random RNA sequence that produces usually, then with its transfection to test cell and monitor the variation of protein expression.Nano-particle of the present invention has two significant advantages that are better than the genome screening technique of these prior aries.At first be that in fact many random siRNA sequences do not have effect, but test cell needs single screening at present, increased the artificial and cost of these experiments of this form.The present invention allows to comprise a plurality of siRNA sequences as the part on the nano-particle, thereby the possibility of accelerating screening speed is provided.Therefore, if in cell, observe effect, then can screen the RNA molecule that exists on the beadlet and determine which sequence to cause this effect by.In addition, because nano-particle provides the delivery system that is used for RNA, so can avoid the use of the transfection reagent that needs in the prior art.This is needed advantage, because transfection reagent self can cause the change of protein expression in the test cell.Therefore, on the other hand in, nano-particle of the present invention can be as the instrument of functional genome, for example such as Nature Reviews, the 3rd volume, in April, 2004 is described in the 318-329 page or leaf.Nano-particle can have each granule more than two, more than 5, more than 10 or more than 20 or more than 100 RNA sequence study in the body gene function and as the instrument of the screening that is used for full genome range.

Aerosol is sent

On the other hand, the invention provides the purposes of nano-particle described herein in aerosol.This is that small size because of nano-particle becomes possible.Aerosol combination can be used for sending the RNA part, particularly is delivered to lung, is used for imaging and/or therapeutic use, for example, infects in the disease of lung in treatment.

In the above-mentioned either side, nano-particle can be connected with therapeutic active substance, such as antibody or tumor-killing medicine.The magnetic properties of nano-particle can also be used for target tumor, by coming guided nano granule to tumor cell with magnetic field.Yet, separately with magnetic field that the nano-particle tumor cell that leads is always unfeasible or accurately, therefore the invention provides a kind of advantage, make nano-particle pass through the tumor-targeting ligands tumor cell that can lead specifically.The probability that this will allow to use less medicine and reduce side effect is because medicine is only for its cell of needs and not for the cell of health.

Another advantage of nano-particle of the present invention is their especially little sizes, and this is so that they are preferably by Cell uptake, even when with targeting or treatment minute sub-connection.

On the other hand, wherein part is that the nano-particle of antigen can be used as vaccine administration, for example, by trajectory, accelerates them and penetrates by the outer field percutaneous of epidermis with sending rifle.Then nano-particle can for example absorb by dendritic cell, along with they by lymphoid migration maturation, cause immunne response adjusting and the antagonism antigen vaccination.

Nano-particle of the present invention can be mixed with the pharmaceutical composition of solid or forms of liquid compositions.Such compositions comprises certain carrier usually, for example solid carrier such as gelatin or auxiliary agent or inert diluent, or liquid-carrier such as water, oil, animal or plant oil, mineral oil or artificial oil.Can comprise normal saline solution, or glycols such as ethylene glycol, propylene glycol or Polyethylene Glycol.Such compositions and preparation contain at least chemical compound of 0.1wt% usually.

Can Nanoparticulate compositions be delivered medicine to the patient by many different approaches.Parenterai administration comprises the administration by following approach: intravenous, and skin or subcutaneous, nose, intramuscular, ophthalmic, through epithelium, intraperitoneal and part (comprise skin, eye, rectum, nose sucks and aerosol), and rectum whole body approach.For intravenous, skin or subcutaneous injection, or in the injection at ailing position, active component will be the acceptable aqueous solution form of non-intestinal, and it is pyrogen-free and has suitable pH, isotonicity and stability.This area relevant technologies personnel can prepare suitable solution fully, the solution of example such as chemical compound or derivatives thereof, and for example, the solution in normal saline is with the dispersion of glycerol, liquid macrogol or oil preparation.

Except one or more chemical compounds, randomly in conjunction with other active component, compositions can also comprise acceptable excipient on one or more materia medicas, carrier, buffer agent, stabilizing agent, isotonic agent, antiseptic or antioxidant or well known to a person skilled in the art other materials.Such material should be nontoxic and the effect of interferon activity composition not.The definite character of carrier or other materials can depend on route of administration, for example, and oral or non-intestinal.

Usually the obtaining liq pharmaceutical composition makes it have approximately 3.0 to 9.0, and more preferably from about 4.5 to 8.5,5.0 to 8.0 pH more preferably from about again.Can be by using buffer agent such as acetate, citrate, phosphate, succinate, Tris or histidine are kept the pH of compositions, and the common scope of application is about 1mM to 50mM.In addition can be by regulating the pH of compositions with the upper acceptable acid of physiology or alkali.

Generally include antiseptic in the pharmaceutical composition and stop growth of microorganism, prolong the storage life of compositions and allow multiple purposes packing.The example of antiseptic comprises phenol, m-cresol, benzylalcohol, p-hydroxy benzoic acid and ester thereof, methyl parahydroxybenzoate, propyl p-hydroxybenzoate, benzalkonium chloride, benzethonium chloride.The common scope of application of antiseptic is approximately 0.1 to 1.0%(w/v).

Preferably, give individuality with prevention effective dose or treatment effective dose (deciding on concrete situation, is treatment although prevention can be looked) with pharmaceutical composition, this is enough to demonstrate the benefit to individuality.Usually, this will cause providing activity useful in the treatment of benefit to individuality.The actual amount of the chemical compound that gives, medicine-feeding rate and time course will depend on character and the order of severity of disease to be treated.The prescription for the treatment of for example, to the decision of dosage etc., is in general practitioner and other internist's responsibilities, and usually considers disease to be treated, the situation of individual patient, site of delivery, other factors that medication and doctor are known.The example of above-mentioned technology and experimental program can be at Handbook of Pharmaceutical Additives(medicated premix handbook), the 2nd edition (editor M.Ash and I.Ash), 2001, (Synapse Information Resources, Inc., Endicott, New York, USA), Remington ' s Pharmaceutical Science(Lei Mingdun pharmaceutical science), the 18th edition, Mack Publishing Company, Easton, Pa., 1990; With Hankbook of Pharmaceutical Excipients(handbook of pharmaceutical excipients), the 2nd edition, find in 1994.For example, preferably with about 0.01 to the 100mg reactive compound of every kg body weight, and more preferably from about the dosage of 0.5 to 10mg/kg body weight delivers medicine to the patient with compositions.

Referring now to accompanying drawing embodiment of the present invention are described for example and not limitationly.

Description of drawings

Fig. 1 has shown the transmission electron micrograph of RNA-Au-Glc nano-particle.

Fig. 2 has shown the existence of testing RNA in the nano-particle that makes.(a) without UV light: 1.RNA-Au-Glc nano-particle+EtBr; 2.Glc-Au+EtBr; 3.Glc-Au; 4. residue+the EtBr of wash solution.(b) use UV light: 1.RNA-Au-Glc nano-particle+EtBr; 2.Glc-Au+EtBr; 3.Glc-Au; 4. residue+the EtBr of wash solution.

Fig. 3 a has shown 48 hours Western blottings from the Her-2/neu albumen of isopyknic SKBR3 cell lysate after the Her-2/neu siRNA transfection.C=contrasts (being untreated) cell; The cell that Au=processes with the siRNA that is attached on the gold nano grain is without RNAiFectamine; S=has the reticent siRNA of RNAiFectamine; NS=has the non-reticent siRNA of RNAiFectamine.

Fig. 3 b has shown 72 hours Western blottings from the Her-2/neu albumen of isopyknic SKBR3 cell lysate after the Her-2/neu siRNA transfection.C=contrasts (being untreated) cell; The cell that Au=processes with the siRNA that is attached on the gold nano grain is without RNAiFectamine.

Fig. 4 has shown the diagram of the preferred nano-particle of the siRNA of comprising of the present invention and carbohydrate ligand.

Fig. 5 shown to siRNA separately (A) or with per 1000 cells, 0.25 μ g(rhombus), 0.5 μ g(is square), 1.0 μ g(triangle), 1.5 μ g(Lycoperdon polymorphum Vitt fork) and 2.0 μ g(black pitch) effect of the cell proliferation of the OVCAR cell of siRNA-nano-particle (B) transfection of siRNA-nano-particle.X-axis=natural law; Y-axis=cell number (log ₁₀).

Fig. 6 shown the effect with the cell proliferation of the OVCAR cell of siRNA-nano-particle transfection, with and without transfection reagent.Used the nano-particle of three kinds of concentration:

1. use transfection reagent (square) and do not use transfection reagent (rhombus);

2. use transfection reagent (Lycoperdon polymorphum Vitt fork) and do not use transfection reagent (triangle);

3. use transfection reagent (circle) and do not use transfection reagent (black fork);

X-axis=natural law; Y-axis=cell number (log ₁₀).

The specific embodiment

The product p185Her-2/neu of Her-2/neu oncogene and coding thereof belongs to epithelial growth factor receptor tyrosine kinase (Bargmann etc., 1986).The HER receptor family is also referred to as Her-1 or erbB-1 by four kinds of cross-film tyrosine kinase: EGFR(), erbB-2(Her-2), erbB-3(Her-3) and erbB-4(Her-4) form.Known Her-2/neu signal pathway (the Yarden ﹠amp that in Growth of Cells and differentiation, vicious transformation and the resistance to chemotherapeutics, plays a crucial role; Sliwkowski, 2001).In approximately 1/3rd people's breast carcinoma or ovarian cancer case, Her-2/neu is overexpression, and its overexpression and poor prognosis relevant (Berchuck etc., 1990).

Having made many trials comes the Her-2/neu in the anticancer to express as potential Therapeutic Method.Humanized monoclonal antibodies (trastuzumab or Trastuzumab) for Her-2/neu is effective (Mendelsohn ﹠amp in Her-2/neu-overexpression metastatic cancer; Baselga, 2000; Baselga etc., 1996) but the expression of Her-3 is raised in discovery.Shown the apoptosis (Roh etc., 2000) of inducing the human breast cancer cell system of overexpression Her-2/neu for the antisense oligonucleotide of Her-2/neu.Use the gene therapy of E1A, send by liposome or by adenovirus vector, can reduce the incidence rate (Chang etc., 1996) that also can reduce the transfer of breast carcinoma model medium and long distance in the Her-2/neu-overexpression ovarian cancer model with the mortality rate of mice with tumor.

Find that the downward modulation that Her-2/neu expresses causes PI3K, the minimizing of Akt and phosphorylation Akt, the expression that it causes cyclin D1 to reduce, cyclin D1 are (the cyclin Sherr ﹠amp of the adjusting of the abortion of a kind of G0/G1 of participation cell and oncogene conversion; Robert, 1999).Relatively antisense oligonucleotide and siRNA effect the latest study proves that siRNA is to making at least 10 times of acceptor gene silences more effective (Miyagishi etc., 2003) on the nM basis.Several researchs have in the past proved that Her-2/neu promotes transcribing of VEGF, and VEGF is effective angiogenic factors (Kumar ﹠amp; Yarmand-Bagheri, 2001), its level significantly reduces after the Her-2/neu expression silencing.Reverse transcription siRNA has improved the level of THBS1 to the downward modulation of Her-2/neu, THBS1 is strong angiogenesis inhibitor (Izumi etc., 2002).Vitro data has proved that HER2siRNA treats the HLAI class surface expression (Choudhury etc., 2004) in the tumor of also significantly upward transferring person.

A. tactful: silence

Her2/Neu cDNA target sequence: AAG CCT CAC AGA GAT CTT GAA

A) justice is arranged: 5 '-G CCU CAC AGA GAU CUU GAAdTdT-3 '

B) antisense: 3 '-dTdTC GGA GUG UCU CUA GAA CUU-5 '

C) justice is arranged: 5 '-G CCU CAC AGA GAU CUU GAAdTdT-3 ' SS

D) antisense: 3 ' SS-dTdTC GGA GUG UCU CUA GAA CUU-5 '

B. annealing

Reticent: 5 ' (a)3 '

3’ （b） 5’

5’ （a） 3’

3’SS （d） 5’

5’ （c） 3’SS

3’ （b） 5’

5’ （a） 3’SS

3’SS （d） 5’

C. possible GNP combination

X=is used in should studying.

D. method

1. cell line

SK-BR-3 breast carcinoma (catalog number (Cat.No.) HTB-30) from ATCC.OVCAR-3 people's ascites adenocarcinoma from NCI-Frederick cancer DCTD tumor/cell line storer (bottle 0502296).

2.siRNA stock solution

The Her-2/neu DNA target sequence of selecting is AAGCCTCACAGAGATCTTGAA.

Have adopted siRNA to have sequence r(GCCUCACAGAGAUCUUGAA) d(TT) MW 7416.25 of 3ThSS(K-salt), antisense sequences r(UUCAAGAUCUCUGUGAGGC) and d(TT) MW 7409.57 of 3ThSS(K-salt) obtain from Qiagen.

The contrast double-stranded sequence of (non-silence) siRNA (catalog number (Cat.No.) 1022076) is from Qiagen, it is r(UUC UCC GAA CGU GUC ACG U that adopted sequence is wherein arranged) d(TT) and antisense sequences be r(ACG UGA CAC GUU CGG AGA A) d(TT), the MW of the K-salt of annealing is 14839.5.

An inclusions (296.65 μ g) that adopted siRNA pipe arranged is dissolved in the 1ml sterile buffer, and (the 2mM magnesium acetate forms 40 μ M liquid storages in pH7.4) for 100mM potassium acetate, 30mM Hepes-KOH.Every μ l contains 0.297 μ g siRNA.

The inclusions (296.38 μ g) of an antisense siRNA pipe is dissolved in the 1ml sterile buffer, and (the 2mM magnesium acetate forms 40 μ M liquid storages in pH7.4) for 100mM potassium acetate, 30mM Hepes-KOH.Every μ l contains 0.296 μ g siRNA.

In order to anneal, with the RNA oligomer solution of 3 μ l and the 5X annealing buffer of 15 μ l merge separately.The concentration of final buffer is 50mM Tris in the water processed of DEPC-, pH7.5-8.0,100mM NaCl.Final volume is 75 μ l, and the final concentration of siRNA duplex is 16 μ M.

With solution incubation 1 minute in 90-95 ℃ water-bath, and make it be cooled to room temperature (that is, being lower than 30 ℃).With the of short duration centrifugal all liq of collecting the pipe bottom of pipe.Slowly cool to room temperature and need 45-60 minute.Resulting solution is stored in-20 ℃ of for subsequent use and tolerance freeze thawing repeatedly.

3.siRNA nanometer gold stock solution

Conventional method

Buy HAuCl from Aldrich chemical company ₄(99.999%) and NaBH ₄The Application standard method is synthesized 2-thio-ethyl-β-D-glucopyranose glycosides in the cur laboratory.For all experiments and solution, use the DEPC(pyrocarbonic acid diethyl ester) nanopure water (18.1m Ω) processed.All microcentrifugal tubes, spatula and bottle are all without the RNA enzyme.

Buy the double-stranded siRNA of annealing from Qiagen-Xeragon Inc.Specification is:

DNA target sequence AAGCCTCACAGAGATCTTGAA.

Adopted siRNA r(GCCUCACAGAGACUUGAA is arranged) d(TT) 3 '-sulfydryl-(SS)-C3-junctional complex on 3 '

(MW 7416.25 of K-salt)

Antisense siRNA r(UUCAAGAUCUCUGUGAGGC) d(TT)

(MW 7409.57 of K-salt)

The preparation of e.RNA-Au-Glc nano-particle

To 100mM, the 2-thio-ethyl-β in the TRIS buffer of pH7.7 (250 μ L)-D-glucopyranose glycosides (0.9mg, 3.75 μ mol) and siRNA(0.148mg, 0.01 μ mol) in the solution, add HAuCl ₄Aqueous solution (22 μ L, 0.025M).Then, with 1N NaBH ₄Aqueous solution (30 μ L) minute several parts of addings, and quick oscillation.Formed brown suspension was vibrated 1 hour in addition at 4 ℃.(AMICON MW 10000,30 minutes, 14000rpm) comes the purification suspension by 4 ℃ by centrifugal filtration.This process is repeated twice, wash with 125 μ L TRIS buffer.Residue in the AMICON filter is dissolved in the TRIS buffer of 250 μ L and lyophilizing, obtains the RNA-Au-Glc nano-particle (resuspension of solid in 1mL water should obtain 6 ± 1 μ M solution of RNA in the 20mMTRIS buffer) of 4mg.Use AMICON(MW3000,4 ℃, 14000rpm) with filtrate desalination and lyophilizing.The weight of residue＜50 μ g.Transmission electron micrograph shown in Fig. 1 (TEM) shows that the particle mean size of granule is 2.8nm, average 807 gold atom/granules, the glucosan derivative of siRNA and 100 molecules as shown in Figure 4 and have an approximate MW 160,000.

F. check the existence of RNA in the nano-particle

The residue that RNA-Au-Glc nano-particle, Glu-Au nano-particle and washing may be contained the RNA-Au-Glc nano-particle of RNA oligonucleotide and glucosan derivative is dissolved in the 30 μ L water separately.With the sample aliquot (1 μ L) of these solution and aqueous solution (1 μ L, the 0.1%v/v) mixing of ethidium bromide (EtBr).Under the UV lamp, observe fluorescence (referring to Fig. 2), proved that the nano-particle of such preparation has the siRNA(Fig. 2 b that mixes, pipe 1), and the nano-particle that only contains glucose does not demonstrate any fluorescence (Fig. 2 b, pipe 2).

4mg is dissolved in 6 ± 1 μ M liquid storages that obtain the 1ml water among the 20mM tris from the siRNA/ nano-Au composite that 148 μ g siRNA produce.Every μ l solution contains the equivalent of 0.078 μ gsiRNA.

Cell is dull and stereotyped to be cultivated

1. in transfection front 24 hours, with 6 * 10 ⁴Individual cell is inhaled to move in the 24 hole flat boards and also with suitable culture medium volume is complemented to 0.5ml.

2. make cell reach 50-80% and converge, probably need 24 hours.

3. remove culture medium and alternative by 300 μ l fresh culture/holes.

With siRNA complex transfectional cell

1. the double-stranded siRNA liquid storage that 3.3 μ l are suitable (or 12.8 μ l nano-Au composites) is dispensed in the 24 hole flat boards that correspondence contains cell.

2. in each hole, add the suitable culture medium of 96.7 μ l (or for nano-Au composite 87.2 μ l) and move 5 times and fully mix by inhaling up and down.

3. in each hole (except the nano-Au composite hole) adds 6 μ lRNAiFect and moves 5 times and fully mix by inhaling up and down.

4. solution was made complex form at room temperature incubation 10-15 minute.

5. be covered on the cell in the 300l culture medium with the suitable transfection composite of 100 μ l.

6. flat board is slightly shaken to mix to avoid vortex.

With flat board at CO ₂In the incubator in 37 ℃ of lower incubation 48-72 hours.

8. remove culture medium and with ice-cold PBS with cell washing three times.

9. with lysis, and measure the protein content of lysate.

10. by the SDS-PAGE isolated protein, then use the Her2/ErbB2 multi-clone rabbit antibody (catalog number (Cat.No.) 2242) of Cell Signalling Technology to carry out western blot analysis.

11. process trace with anti-rabbit igg-HRP conjugate, then ECL colour developing.

G. result

Shown the Preliminary Observational Results that uses 1 μ g siRNA/ hole among Fig. 3 a and the 3b.The siRNA-gold nano grain is added in the cell RNAiFectamine useless.The SKBR3 cell reach 80% converge slower than OVCAR cell.SKBR 3 results from transfection after 48 hours lysate and OVCAR result from transfection after 72 hours lysate.The schematic diagram of nano-particle is shown among Fig. 4.

The siRNA-Au-Glc nano-particle is to the cell avirulence

Separately with siRNA with use the siRNA that puts together with the sugared nano-particle of gold to come transfectional cell.Fig. 5 has shown that the siRNA that nano-particle is puted together is effective and free of toxic effects.Observe the dose-dependent effects to cell number, show that the siRNA nano-particle has improved cell proliferation.

The SiRNA-Au-Glc nano-particle enters cell

With siRNA-nano-particle transfection OVCAR cell, with or need not usually use the needed transfection reagent of siRNA transfectional cell.Fig. 6 has shown that entering cell for the siRNA-nano-particle does not need transfection reagent.The result shows that the siRNA nano-particle is delivered in the cell effectively, even in the non-existent situation of transfection reagent; In fact, transfection reagent useless send it seems more effective.The dose-dependent effects of cell number has been shown real response to the siRNA-nano-particle.

List of references

List of references all specially is incorporated herein by reference referred in this.

Pal-Bhadra etc., RNAi-Mediated targeting of Heterochromatin by the RITS complex(is by the heterochromatin targeting of the RNAi-mediation of RITS complex) .Science (2004) vol.303,669.

Verdel etc., heterochromatin silence and HP1 location depend on RNAi mechanism in the Heterochromatic silencing and HP1 localization in Drosphila are dependent on the RNAi Machinery(fruit bat) .Science (2004) vol.303,672.

Elbashir etc., the RNA in the mammalian cell that the duplex mediation of the RNA of cells(21 nucleotide of Duplexes of 21-nucleotide RNAs mediate RNA interference in cultured mammalian is cultivated disturbs) .Nature 2001; 411:494-8.

Tuschl, RNA interference and small interfering RNAs(RNA disturbs and siRNA) .Chem Biochem 2001; 2:239-45.

Bargmann etc., The neu oncogene encodes an epidermal growth factor receptor-related protein(neu oncogene coding schedule skin growth factor receptor associated protein(RAP)) .(1986) Nature 319,226-230.

Yarden ﹠amp; Sliwkowski, Untangling the ErbB signalling network (untiing ErbB signal net) .(2001) Nat Rev Mol Cell Biol 2,127-137.

Berchuck etc., the poor survival of the overexpression of Overexpression of HER-2/neu is associated with poor survival in advanced epithelial ovarian cancer(HER-2/neu and advanced epithelial ovarian carcinoma is relevant) .(1990) Cancer Res 50,4087-4091.

Mendelsohn ﹠amp; Baselga, The EGF receptor family as targets for cancer therapy(is as the EGF receptor family for the treatment of of cancer target) .(2000) Oncogene 19,6550-6565.

Baselga etc., Phase II study of weekly intravenous recombinant humanized anti-p185HER2 monoclonal antibody in patients with HER2/neu over expressing metastatic breast cancer(have that the II phase of the anti-p185HER2 monoclonal antibody of intravenous recombinant humanized is studied weekly among the HER2/neu patient of overexpression transitivity breast carcinoma) .J Clin Oncol 1996; 14:737-44.

Roh etc., the human cancer cell apoptosis that overexpression ER2/neu is induced in the downward modulation that Down regulation of HER2/neu expression induces apoptosis in human cancer cells that over-express HER2/neu(HER2/neu expresses) .(2000) Cancer Res 60,560-565.

Chang etc., Inhibition of intratracheal lung cancer development by systemic delivery of E1A(suppresses pulmonary carcinoma development in the trachea by systemic delivery E1A) (1996) Oncogene 13,1405-1412.

Sherr ﹠amp; Roberts, CDK inhibitors:positive and negative regulators of Gl-phase progression(CDK inhibitor: the positive and negative instrumentality that the G1-phase makes progress) .(1999) Genes Dev 13,1501-1512.

Miyagishi etc., Comparison of the suppressive effects of antisense oligonucleotides and siRNAs directed against the same targets in mammalian cells(antisense oligonucleotide and comparison for the inhibition of the siRNA of identical target in the mammalian cell) .Antisense Nucleic Acid Drug Dev 2003; 13:1-7.

Kumar ﹠amp; Yarmand-Bagheri, the effect of The role of HER2 in angiogenesis(HER2 in angiogenesis) .(2001) Semin Oncol 28,27-32.

Izumi etc., Tumour biology:herceptin acts as an antiangiogenic cocktail(oncobiology: Trastuzumab is as the mixture of angiogenesis inhibitor) .(2002) Nature 416,279-280.

Choudhury etc., Small interfering RNA (siRNA) inhibits the expression of the Her2/Neu gene, upregulates HLA Class I and induces apoptosis of Her2/Neu positive tumour cell lines(siRNA (siRNA) suppresses the expression of Her2/Neu gene, the apoptosis that raises I class HLA and induce Her2/Neu positive tumor cell system) .Int J Cancer 108,71-77,2004.

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WO 02/32404, enjoys the PCT application of GB-A-0313259.4 priority.

Claims (10)

1. the purposes of the nano-particle that contains core in the medicine of the disease of being alleviated by the downward modulation of gene expression for the preparation for the treatment of or the disease relevant with the overexpression of gene, described core comprises metal and/or semiconductor atom, wherein said core and a plurality of part are covalently bound, this part comprises (i), and at least one contains the part of carbohydrate group and (ii) at least one RNA part, this RNA part comprises siRNA, shRNA or miRNA molecule, wherein said RNA part targeting is also reduced described gene, and wherein said nano-particle is used in the situation that lack the transfection agents administration.

2. the purposes of claim 1, wherein said disease is cancer, viral infection or eyes degeneration of macula.

3. the purposes of claim 2, wherein said cancer is that breast carcinoma or described virus are HIV, hepatitis or influenza.

4. the purposes of claim 1, wherein said nano-particle and RNA part are covalently bound by linking group.

5. the purposes of claim 4, wherein said linking group is mercapto groups, ethylidene group or peptide group.

6. each purposes of aforementioned claim, wherein said part is siRNA part and the 3 ' jag that comprises 2 ribonucleotides.

7. the purposes of claim 1, wherein the first sense strand of RNA molecule is covalently bound with the nano-particle core by its 3 ' end.

8. the purposes of claim 1, wherein said RNA part is based on the Her2 gene order.

9. the purposes of claim 1, the core of wherein said nano-particle comprises gold atom and has 0.5 to 10nm average diameter.

10. claim 1 or 8 purposes, wherein said RNA part has following:

Between 10 to 40 ribonucleotides,

Between 17 to 30 ribonucleotides,

Between 19 to 25 ribonucleotides, or

Between 21 to 23 ribonucleotides.

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